凝血酶敏感蛋白區域包含蛋白7A經由αvβ3整合素-樁蛋白相關之訊號傳遞路徑調控血管內皮層細胞之移動

Thrombospondin Type I Domain Containing 7A Modulates Cell Motility in Endothelial Cell through αvβ3 Integrin-Paxillin Related Signaling Pathway

蘇佩慈(Pei-Tsu Su)

指導教授 ： 莊永仁

國立清華大學/生命科學院/生物資訊與結構生物研究所/碩士(2009年)

摘要

凝血酶敏感蛋白區域包含蛋白7A (THSD7A)是個新穎的蛋白質，會在血管新生的過程中調控血管內皮層細胞的移動。生物資訊的分析結果預測THSD7A是一個膜蛋白，包含了十一個凝血酶敏感蛋白重複區域(thrombospondin-type-I repeats, TSR)，一個CD36結合區域，以及一個RGD序列。這些特徵暗示THSD7A可能參與了細胞移動和細胞與胞外基質的交互作用。我們先前的研究顯示，THSD7A會調控人類臍帶靜脈內皮層細胞(HUVEC)的移動，以及斑馬魚發育時期體節間血管的新生。然而，對於THSD7A引導血管生成的下游調控機制仍尚未釐清。由於THSD7A被預測為一個膜蛋白，首先我們希望利用細胞免疫螢光染色技術來探討THSD7A在HUVEC中的表現位置。我們發現THSD7A表現在細胞核周圍，意味著THSD7A在內質網和高基氏體被重新合成以及運送。除此之外，THSD7A也會與avb3整合素(integrin)以及樁蛋白(paxillin)一起聚集在位於絲狀肌動蛋白束末端(actin filament)的集中附著點(focal adhesion sites)上。綜合上述，這些發現暗示著THSD7A可能是一個藉由內質網和高基氏體運輸到細胞膜的集中附著點蛋白。THSD7A有一段RGD氨基酸序列，這種序列被廣為知道能與integrin結合並幫助其活化。在integrin活化後，一個被稱為paxillin的集中附著點相關輔助蛋白，已知在細胞移動的機制中扮演關鍵性的角色。因此我們假設THSD7A可能經由integrin－paxillin的路徑來調控細胞的移動。我們發現在細胞產生方向性移動的狀態下，THSD7A會聚集在細胞的前端，這與paxillin在細胞移動時的分佈情形相同。我們接著利用免疫共沈澱法證明THSD7A會與paxillin結合。除此之外，抑制THSD7A在HUVEC中的表現時，會導致HUVEC產生較寬的層狀偽足(lamellipodia)以及actin聚集的現象，這個發現類似於其他研究指出的有功能性缺陷的paxillin在細胞中所造成的影響。這些結果顯示THSD7A可能是一個新的paxillin結合蛋白，並且可能藉由integrin-paxillin所調控的細胞骨架重組機制來影響細胞的移動。綜合以上所述，我們的研究結果顯示THSD7A對於血管內皮層細胞除了細胞移動外尚有更廣效性的影響。對於THSD7A更進一步的研究將會對於內皮細胞生理上的機制以及病理上的生長失序有更多的瞭解。

關鍵字

凝血酶敏感蛋白區域包含蛋白7A ； 樁蛋白 ； avb3整合素 ； 細胞移動 ； 血管內皮層細胞

並列摘要

Thrombospondin Type I Domain Containing 7A (THSD7A) is a novel protein that mediates endothelial cell migration in angiogenesis. Bioinformatic analysis predicted THSD7A to be a membrane protein containing eleven thrombospondin-type-I repeats (TSR), one CD36-binding domain, and one RGD motif. These features imply THSD7A may be involved in cell migration and cell-to-ECM interaction. From previous studies, we have learned THSD7A regulates human umbilical vein endothelial cells (HUVECs) migration in vitro and affects zebrafish intersegmental vessels (ISV) angiogenesis during its development in vivo. However, the underlying mechanism by which THSD7A affects vessel guidance and patterning remains unclear. Since THSD7A was predicted to be a membrane protein, we first performed immunocytochemistry analysis to examine the subcellular localization of endogenous THSD7A in HUVEC. We found that THSD7A could be detected at peripheral nucleus area, which suggests its de novo synthesis and processing in the endoplasmic reticulum (ER) and Golgi apparatus. In addition, THSD7A could also be found at focal adhesion sites that co-localized with avb3 integrin and paxillin at the extremities of actin cytoskeleton in HUVECs. Taken together, these findings implied THSD7A may be a focal adhesion protein that was transported to membrane through ER-Golgi. THSD7A has a RGD motif, which is well known to interact with integrins and to activate them. Upon integrin activation, a focal adhesion-associated adaptor protein called paxillin is known to play as a key role in regulating cell motility. Therefore, we hypothesized that THSD7A may also regulate cell motility through the integrin-paxillin pathway. We found that THSD7A can be observed at the leading front of directed migrating cells, and THSD7A was similarly distributed as paxillin in migrating cells. We then verified the physical interaction of THSD7A and paxillin by co-immunoprecipitation assay. Furthermore, THSD7A knockdown in HUVECs induced the formation of broad lamellipodia-like structures and actin condensation, which are similar to paxillin-deficient cells observed in other studies. Altogether, these findings suggested THSD7A is a new binding partner of paxillin and it may mediate cell migration through integrin-paxillin-associated cytoskeletal reorganization. In summary, our studies revealed THSD7A has a wide range of effects beyond cell migration on primary endothelial cells. Further analysis of THSD7A will shed light on physiological mechanisms of endothelial cells, and may provide new insights into relevant pathological disorders.

並列關鍵字

THSD7A ； paxillin ； alphavbeta3 integrin ； cell migration ； endothelial cell

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